The invention relates to general measuring systems in the sector of analytical laboratory technology, in which microfluidic microchips are used for the chemical, physical, and/or biological analysis or synthesis of substances, which feature a channel structure by means of which the substances, with the imposition of a physical potential, and of an electrical or hydraulic potential in particular, are capable of movement in accordance with the channel structure. The invention relates in this context, in particular, to the injection of substances into such a microfluidic microchip.
A microchip of the type described in the preamble, and a corresponding microchip laboratory system, are described, for example, in U.S. Pat. No. 5,858,195. In a microchip of this kind, the substances concerned are moved by means of a system of channels connected to one another and integrated on the microchip. The movement of these substances in these channels is controlled by means of electrical fields, which are imposed along these transport channels. Because of the high-precision control of the substance movement which this makes possible, and the very exact metering capacity of the substance masses moved in each case, the substances can be mixed precisely with regard to the stoichiometry required, can be divided, and/or can induce chemical or physical-chemical reactions. With this microchip, the channels provided for in the integrated structural design feature a plurality of substance reservoirs, which contain the substances required for the chemical analysis or synthesis. The movement of the substances from these reservoirs along the transport channels is effected in this case by electrical potential differences. The substances moving along the transport channels accordingly come in contact with different chemical or physical environments, which then make possible the required chemical or chemical-physical reactions between the individual substances. In particular, the microchip features one or more crossings between the transport channels, in which the intermixing of the substances takes place. By the simultaneous use of different electrical potentials on the different substance reservoirs, it becomes possible for the volume flows of the different substances to be selectively controllable by means of one or more crossing points, and therefore, solely on the basis of the electrical potentials applied, for a precise stoichiormetric yield to be effected.
The movement of the substances by means of electrical high voltage in this situation, however, represents only one variation. For example, it is possible for the potential difference required for the movement of the substances also to be implemented by means of imposing a pressure means on the substances, for preference a suitable gas means such as a noble gas. The movement of the substances can also be effected by the use of a suitable temperature profile, in which situation the movement takes place due to the thermal expansion of the particular substance. The selection of the individual means for the provision of a potential or of a force for the movement of the substances on the microchip is based in this case in particular on the physical properties inherent in the individual substances. In the case of substances with charged particles, such as charged or ionized molecules or ions, the movement of the substances is effected for preference by means of an electrical or electromagnetic field of suitable strength. The path which the substances pass over in each case is calculated in this situation in particular on the basis of the field strength and the duration of the field applied. The movement of the substances is effected in this case either etectrokinetically, in other words, in the case of charged substances, due to the effect of the external electrical field on the charge which is present in each case. As an altemative, or in parallel with this, the movement of the substance can be effected by electro-osmosis in the case of substances (particles) in solution in solvents, in which situation the solvent is essentially moved on the basis of a charged double layer being formed with many materials, such as glass, on the surface which is adjacent to an electrolyte, and accordingly incurring a net counter-current of the substances. In the case of substances which are free of an electrical charge, however, or which are not in solution, the movement of the substances is effected in most cases with the aid of what is referred to as a flow means.
The microfluidic microchips described are therefore particularly characterised by the fact that, because of the very small dimensions of the transport channels on the microchip, only relatively small volumes of substance are moved, in the range from picolitres to nanolitres. An analysis or synthesis of small substance volumes of this type therefore also implies extremely high-resolution detection devices for the measurement of these small substance masses. The measurement resolution of such measuring devices is therefore largely determined by the sensitivity of the detector used in each case, as well as the underground noise caused by the measurement arrangement as a whole.
It is known that the signal-to-noise ratio of the measuring devices concerned in this case can in principle be substantially improved by the tests which are to be conducted in each case being carried out several times, and by mean values then being formed from the measuring results obtained. With this procedure, however, it must be borne in mind that certain tolerances are to be maintained between the repeated measurement cycles, in order in particular to avoid the mixing of substances or the superimposition of the measurement signals detected. These dead periods and the pulse widths in each case for the injection of substances therefore also determine, conversely, the minimum duration of a measuring cycle, and therefore also directly determine the limits of the signal-to-noise ratios which can in principle be achieved.
In addition to this, the measurement cycle can also in principle be shortened by multiple substance injection during an analysis attempt, in particular when substance separation is being carried out. This has the disadvantage, however, that the measurement signals which are superimposed in this process must subsequently be relatively laboriously disentangled. It is often also not possible to carry out subsequent disentanglement of the measurement signals with sufficient precision.
The object underlying the invention is to provide a microfluidic microchip of the type concerned, as well as a process for its operation, with which, despite the physical and technical restrictions described heretofore, the signal-to-noise ratio can be improved in comparison with the pertinent prior art during the performance of the tests described.
A further object involves achieving such improvement withoutstantial volumes of the substance to be processed being required, or additional costs being required for the manufacture of such microchips.
These objectives are achieved according to the invention by the features of the independent claims. Preferred and advantageous embodiments of the invention are described in the dependent claims.
According to a first variant of the microchip according to the invention, provision is made in particular for the fact that, in a first operating cycle or a first operating phase, a leading channel or leading channel section can be filled with a continuous substance flow by the imposition of a constant potential on a feed channel and a discharge channel, whereby the individual substance components, in particular the slowest moving substance components, extend continuously and homogenously along the leading channel. Once a continuous volume flow of substance of this kind has been formed, the substance volume contained in the leading channel can to advantage be guided out of the leading channel by switching over the potential, and can be injected into a channel provided for the conduct of the test.
By means of this type of substance injection, spatially precisely defined substance and volume units can to advantage be generated. By the use of an appropriately modulated potential, these substance-volume units can be conducted out of the leading channel section. By the use of a specified sequence of pulses, the appropriate substance volume sequences can then be generated.
The proposed channel structure can for preference be operated in two operational cycles or phases, in which situation it is possible to switch over between the two operating cycles by means of switching means. In a first operating cycle, the substances are continuously conducted into the leading channel section, and in the second operating cycle are then lead out of the channel section by the application of said modulated potential.
The generation of even more complex substance volume sequences can be achieved by making provision for at least two leading channel sections. This makes it possible for substance volume units of different lengths, in particular of a multiple of a unit length, to be generated in one operating cycle. For example, the substance volume units contained in two leading channel sections arranged next to one another can be guided out of this leading channel section by the application in one operating section of a suitable potential, as a result of which a substance volume unit is generated of twice the length of the unit length defined by the individual leading channel sections.
According to a further embodiment, a substance-conducting buffer reservoir connected to the leading channel is provided for the intermediate storage of substance volume units which have already been generated. With this embodiment, the substance volume units are accordingly first conducted to the buffer reservoir and only then conducted out of this to a channel provided for the actual performance of the test. To carry out the test, it is possible in this situation in particular to provide for a substance-conducting separation channel connected to the leading channel and the buffer reservoir respectively. To accommodate substances in the separation channel which have already been subjected to potential it is possible to make provision to advantage of a collecting reservoir connected to the separation channel in such a way as to conduct the substance.
The microchip proposed according to the invention can also be designed in such a way that, by means of a first and second activation means a potential of a different type in each case can be provided, in particular an electrical potential with the first activation means and a mechanical (hydraulic) potential with the second activation means, or vice-versa.
In a second variant, the microchip according to the invention features in particular a channel structure with a feed/discharge channel for feeding/discharging the substances, which is connected in a substance-conducting manner at a crossing point with a separation channel for carrying out the chemical analysis or synthesis. In order to build up a potential for the continuous movement of the substances in the feed/discharge channel, provision is made in this situation for first activation means. In addition, second activation means are provided for creating a temporally-modulated or amplitude-induced potential, by means of which the substances located in the separation channel are capable of modular movement, which is turn allows for the formation of the substance volume units separated accordingly by the modulated potential. With this embodiment of the channel structure, by contrast with the first variant, the substance volume units are generated in intersecting transport channels by means of suitable activation means. This variant is based on the concept of conducting a continuous volume flow through such a channel intersection, and, in a second operational phase, switching over the potential required for the movement of the substances for a specified period of time, in such a way that the continuous volume flow is diverted during the specified period of time via a further transport channel. By re-establishing the potential state which prevailed in the first operational phase, it is then possible to generate overall substance volume units of adjustable sizes. The concept on which this variant is based consists to a certain extent of providing the substance volume units in particular by means of an electrically switchable set of channel points. This variant accordingly requires in principle only minor changes to the channel structure in comparison with the first variant.
It is emphasised that both variants feature the advantage, in comparison with the prior art that the constant and continuous volume flow generated in the first operating phase leads to advantage to a situation in which even relatively slow moving substance components can extend or spread over the entire volume of the individual volume unit, and therefore reproducibly mixed and extremely homogenous substance mixtures can be injected as substance volume units into a separation channel.
In addition to this, both variants have the advantage in common that the individual injectors in each case are capable of being controlled exclusively by the application of an electrical potential, and an electrical voltage in particular, and accordingly require no moving parts at all.
In addition to this, with the proposed microchip it is possible for extremely readily reproducible volume units to be generated for the injection, whereby the standard deviation is approximately in the range of a few percent. This very precise production of injection volumes, together with the relatively low dead times or response times can be applied to advantage for the generation of predetermined injection sequences or substance/volume sequences. In particular, with such microchips, the randomly-scattered substance volume sequences described in detail hereinafter can be generated for injection into a separation channel of such a microchip.
It is self-explanatory that provision can also be made with the second variant for the switchover between the two operating phases to be automated by means of suitable switching means.
The invention further relates to a measurement device and a process for the operation of a microfluidic microchip of the type described heretofore. With the measuring device according to the invention, provision is made in particular for generator means for the creation of the constant potential required in the first operating phase, as well as foe the production of the modulated signal (potential) required in the second operating phase, which can in particular be arranged combined in one single signal generator. For preference, the switchover between the two operating phases is capable of being automated by means of suitable switching means.
To generate a temporally alternating potential, provision can also be made for a signal generator which generates pulsed signals with essentially constant amplitude and variable oscillation duration and oscillation phase. The signal generator may in particular be designed as a square-wave generator. A square-wave generator allows in particular for the production of normed substance volume flows, and therefore of correspondingly precisely adjustable substance volume units.
According to a particularly advantageous further embodiment of the device according to the invention, provision can be made for the generator means for the production of the temporally or amplitude altemating potential to be triggered by means of pseudo-randomly scattered binary digit sequences created by a processor unit. These binary digit sequences may in this situation represent, in particular, binary bit sequences similar to Hadamard sequences. This allows for the measured spectra to be converted into an electropherogram by means of a cross-correlation analysis or calculation of the spectra measured in each case, by making recourse to the injected substance volume flows and the resultant measurement signals. The signal-tonoise ratio of the resultant electropherogram in particular is then substantially improved, as explained in detail hereinafter.